Purpose: To assess tobacco, alcohol, cannabis and benzodiazepine use in methadone maintenance treatment (MMT) as potential sources of variability in methadone pharmacokinetics. Methods: Trough plasma (R)- and (S)-methadone concentrations were measured on 77 Australian and 74 Swiss MMT patients with no additional medications other than benzodiazepines. Simple and multiple regression analyses were performed for the primary metric, plasma methadone concentration/dose. Results: Cannabis and methadone dose were significantly associated with lower 24-h plasma (R)- and (S)-methadone concentrations/dose. The models containing these variables explained 14–16% and 17–25% of the variation in (R)- and (S)-methadone concentration/dose, respectively. Analysis of 61 patients using only CYP3A4 metabolised benzodiazepines showed this class to be associated with higher (R)-concentration/dose, which is consistent with a potential competitive inhibition of CYP3A4. Conclusion: Cannabis use and higher methadone doses in MMT could in part be a response to — or a cause of — more rapid methadone clearance. The effects of cannabis and benzodiazepines should be controlled for in future studies on methadone pharmacokinetics in MMT.